A Link between Meiotic Prophase Progression and Crossover Control

During meiosis, most organisms ensure that homologous chromosomes undergo at least one exchange of DNA, or crossover, to link chromosomes together and accomplish proper segregation. How each chromosome receives a minimum of one crossover is unknown. During early meiosis in Caenorhabditis elegans and many other species, chromosomes adopt a polarized organization within the nucleus, which normally disappears upon completion of homolog synapsis. Mutations that impair synapsis even between a single pair of chromosomes in C. elegans delay this nuclear reorganization. We quantified this delay by developing a classification scheme for discrete stages of meiosis. Immunofluorescence localization of RAD-51 protein revealed that delayed meiotic cells also contained persistent recombination intermediates. Through genetic analysis, we found that this cytological delay in meiotic progression requires double-strand breaks and the function of the crossover-promoting heteroduplex HIM-14 (Msh4) and MSH-5. Failure of X chromosome synapsis also resulted in impaired crossover control on autosomes, which may result from greater numbers and persistence of recombination intermediates in the delayed nuclei. We conclude that maturation of recombination events on chromosomes promotes meiotic progression, and is coupled to the regulation of crossover number and placement. Our results have broad implications for the interpretation of meiotic mutants, as we have shown that asynapsis of a single chromosome pair can exert global effects on meiotic progression and recombination frequency

Serine integrase chimeras with activity in E. coli and HeLa cells

In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC) and phiC31-TG1 (CT) hybrid integrases are active in E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{−1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies

Progression from Early to Late Pachytene Is Delayed in X Asynapsis Mutants

<div><p>(A) Gonads imaged at 100× and composited. Top: N2; bottom: <i>him-8(mn253)</i>. The three yellow lines demarcate a rough separation of the gonad into four sections from left to right: premeiotic, transition zone, early pachytene, and late pachytene. Although each zone contains mainly nuclei of one meiotic substage, the transitions are not completely abrupt, necessitating counting of all nuclei in the gonad to obtain accurate staging. The <i>him-8</i> gonad contains a higher proportion of early pachytene nuclei, and a lower proportion of late pachytene nuclei, than the N2 gonad. Inset: synapsis is complete between autosomes in the early pachytene region in <i>him-8</i> gonads, whereas X chromosomes do not synapse (chromosomes, stained with DAPI, are displayed in red; the central element protein SYP-1, detected with immunofluorescence, is displayed in green; arrowheads mark the pycnotic X chromosomes which do exhibit SYP-1 staining.) Right: graph displaying raw numbers of nuclei at each substage for N2 and <i>him-8</i> gonads. Total numbers of nuclei are displayed for four (N2) or five <i>(him-8)</i> gonads. P, premeiotic; TZ, transition zone; EP, early pachytene, LP, late pachytene.</p><p>(B) High-magnification view of transitions between meiotic prophase substages. Nuclei are tinted to highlight the classification of stage: green, transition zone nuclei; orange, early pachytene; blue, late pachytene. Arrowheads indicate exemplars of each stage, also shown from left to right in the inset (upper right).</p><p>Scale bar, 50 μm.</p></div

Crossover Alteration in X Chromosome Asynapsis Backgrounds

<div><p>Two genotypes <i>(him-8, meDf2)</i> were assayed for recombination by genetic crossing and SNP mapping. (A) The genetic distance between two visible markers on chromosome III was assayed by genetic crosses. Map distance increased from 17 centimorgans in N2, to 29 centimorgans in both <i>him-8</i> and <i>meDf2</i>.</p><p>(B) Single-nucleotide polymorphism mapping of chromosomes II, III, and V. Five SNP markers were used, resulting in four intervals across the chromosome in which recombination could be assayed (<i>x</i> axis). The relative physical length of each region is shown by the distance between gray bars in the graph background.</p><p>(C) Physical and genetic locations of single-nucleotide polymorphisms analyzed. The horizontal bars represent the physical length of the chromosomes (II, III, and V), with polymorphisms indicated above, proportional to their physical distance. Below each bar the polymorphisms are traced to a horizontal dashed line representing the interpolated genetic distance between them, also indicated numerically in centimorgans. Labels for each interval, numbered 1–12, correspond between B and C.</p></div

Progression of RAD-51 Focus Formation and Removal in Wild-Type and Meiotic Mutants

<div><p>(A) From top to bottom, gonads from wild-type (N2), <i>him-8, msh-5,</i> and <i>him-8 msh-5</i> worms are shown. In wild-type (top), RAD-51 foci appear in the transition zone and disappear in early pachytene. In all mutant conditions, RAD-51 focus formation begins in the transition zone, but persists throughout early pachytene, only disappearing at the very end of the gonad in late pachytene. Scale bar, 50 μm.</p><p>(B) Quantitation of RAD-51 focus formation in wild-type and mutant conditions. Gonads were automatically divided into six equally sized regions, and nuclei assigned to each region based on their location. Graphs display box-whisker plots of focus numbers. The <i>x</i> axis indicates bins of equal length along the gonad; the <i>y</i> axis indicates the number of RAD-51 foci observed in a nucleus. The center horizontal line of each box indicates the median value; the box top and bottom indicate the first and third quartile values; the lines above and below the boxes extend to the entire range of measurements. Number of nuclei observed for each case are indicated at upper right.</p></div

Long-Term Expression of Human Coagulation Factor VIII in a Tolerant Mouse Model Using the phi C31 Integrase System

We generated a mouse model for hemophilia A that combines a homozygous knockout for murine factor VIII (FVIII) and a homozygous addition of a mutant human FVIII (hFVIII). The resulting mouse, having no detectable FVIII protein or activity and tolerant to hFVIII, is useful for evaluating FVIII gene-therapy protocols. This model was used to develop an effective gene-therapy strategy using the phi C31 integrase to mediate permanent genomic integration of an hFVIII cDNA deleted for the B-domain. Various plasmids encoding phi C31 integrase and hFVIII were delivered to the livers of these mice by using hydrodynamic tail-vein injection. Longterm expression of therapeutic levels of hFVIII was observed over a 6-month time course when an intron was included in the hFVIII expression cassette and wild-type phi C31 integrase was used. A second dose of the hFVIII and integrase plasmids resulted in higher long-term hFVIII levels, indicating that incremental doses were beneficial and that a second dose of phi C31 integrase was tolerated. We observed a significant decrease in the bleeding time after a tail-clip challenge in mice treated with plasmids expressing hFVIII and phi C31 integrase. Genomic integration of the hFVIII expression plasmid was demonstrated by junction PCR at a known hotspot for integration in mouse liver. The phi C31 integrase system provided a nonviral method to achieve long-term FVIII gene therapy in a relevant mouse model of hemophilia

Recombinase-Mediated Reprogramming and Dystrophin Gene Addition in <i>mdx</i> Mouse Induced Pluripotent Stem Cells

<div><p>A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the <i>mdx</i> mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram <i>mdx</i> fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies.</p></div

Pluripotency of <i>mdx</i> iPSC.

<p>(a) Reprogrammed <i>mdx</i> iPSC colony W9; bright field, GFP, and alkaline phosphastase staining. (b) GFP fluorescence and immunofluorescence staining of Oct4, Sox2, Nanog, and SSEA-1 in W9 iPSC before and after (W987) Cre-mediated excision of reprogramming genes and in mESC. Scale bar  = 50 µm. (c) Quantitative RT-PCR data showing expression of Oct4, Sox2, Nanog, and c-Myc in W9 and W987 iPSC, as well as in mESC controls and in the parental <i>mdx</i> adult fibroblasts. (d) Promoter methylation status of Oct4 in W9 and W987 iPSC and in mESC and adult fibroblast controls. Five different CpG islands were analyzed, indicated by their distance from the transcription start site. Open circles reflect low methylation (0–25%), gray circles represent medium (26–75%), and black circles indicate high (76–100%) methylation. (e) Embryoid bodies grown from W987 iPSC and stained for markers of the three germ layers. Day 14 embryoid bodies were stained with antibodies against smooth muscle actin (SMA), α-fetoprotein (AFP), and βIII-tubulin (Tuj1), indicating mesodermal, endodermal and ectodermal differentiation <i>in vitro</i>, respectively. DAPI was used to stain the nuclei. Alexa 594-labeled secondary antibodies were used. (f) Chromosome counts were performed in the parental <i>mdx</i> adult fibroblasts and in iPSC before (W9) and after (W987) Cre excision. The normal murine chromosome number of 40 was observed.</p

Genome engineering.

<p>(a) Southern blot analysis. iPSC clones derived from <i>mdx</i> fibroblasts and reprogrammed using pCOBLW and pVI were probed with a sequence from the EGFP gene. The number of bands indicated the number of copies of the reprogramming plasmid that integrated. W3, W4, W5, W10, and W9 represented a subset of the reprogrammed colonies that were screened. (b) Bxb1-mediated site-specific integration of therapeutic plasmid. Puromycin-resistant colonies were picked. (c) Representative subclones W9D1–W9D10 were analyzed by PCR to detect the expected <i>attR</i> (591 bp) and <i>attL</i> (431 bp) Bxb1 junction bands. (d) Left: Cre-mediated excision of unwanted sequences causes a loss of GFP fluorescence. Cre resolvase was introduced in representative subclones W9D8 and W9D10 to excise the reprogramming cassette and other sequences no longer needed. After excision, GFP expression was extinguished in colonies. Right: Representative subclones W9D8E3, W9D8E4, W9D8E7 (W987), W9D8E8, W9D8E17, and W9D10E1 were analyzed by nested PCR to detect the expected <i>loxP</i> 167 bp junction fragment indicative of successful excision.</p

Myofiber differentiation and engraftment.

<p>(a) Immunofluorescence staining of dystrophin in W9, W987, and ESC. Myosin heavy chain (MHC) identified muscle cells after differentiation. DAPI was used to stain nuclei. (b) Myotube formation in differentiated W987 and W9 iPSC and ESC. Note that W9 iPSC did not form myotubes. (c) Engraftment of corrected <i>mdx</i> iPSC in mouse TA muscle. Approximately 750,000 W987 iPSC that were differentiated for 13 days <i>in vitro</i> and sorted for the SM/C-2.6 antibody were injected into the TA muscle of an irradiated <i>mdx/;SCID</i> mouse. After three weeks, muscle sections were prepared and stained. Staining for laminin delineated individual muscle fibers, while staining for dystrophin revealed engraftment of corrected iPSC (arrows). (d) Numbers of dystrophin-positive fibers per TA muscle, total section, are shown for engrafted muscle versus uninjected contralateral muscle (control).</p